Steroid receptor analyses are performed in the clinical environment with increasing frequency due, at least, in part to the heightened awareness for the diagnostic value of the results. Specifically, it is now becoming generally accepted that an assessment of the presence of steroid receptors in biopsies of human breast carcinomas is essential to enable correct selection of hormone therapy. Although the mechanisms regarding interaction between receptors and therapy is as yet undetermined, it does seem clear that both estrogen and progesterone receptors may be utilized as predictive indices of a breast cancer patient's response to hormonal manipulation. Indeed, it is a commonly accepted principle that the presence of both receptors enhances the effectiveness of steroid therapy and makes such a route of treatment a viable alternative vis-a-vis chemotherapy or surgery. The chart presented below characterizes the present view regarding the presence or absence of estrogen receptors (ER) and/or progesterone receptors (PgR): ##STR1##
A general but informative discussion is provided by Wittliff, et al., Physiopathology of Endocrine Diseases and Mechanisms of Hormone Action, pages 397-411, 1981, Alan R. Liss, Inc., New York, New York, in a chapter entitled "Methods of Steroid Receptor Analyses and Their Quality Control in the Clinical Laboratory."
The progress in developing ER and PgR tests to clinical significance has been impeded by a variety of factors acting in concert. Foremost of those is the basic instability of steroid receptors due to their heat labile nature. This is of particular concern with control reagents necessary for comparison with any tissue biopsy. Consequently, the conventional art has sought a variety of mechanisms in an attempt to ammeliorate the stability problem.
Relatively early it was discovered that the progesterone and estrogen receptors were relatively stable while in the form of tissue preparations, particularly when such preparations were lyophilized and kept frozen. See for instance Koenders, et al., "Influence of Lyophilization and Subsequent Storage of Target Tissue on Steroid Receptors, Steroid Receptors and Hormone-Dependent Neoplasia" edited by Wittliff, Masson Publishing, New York, N.Y. Such a solid tissue control material is comparable to patient biopsy tissue and accordingly must be treated in like fashion. Some investigators advocate the tissue control's merit as providing a control for the homogenization aspects of the assay procedure. To date, only one tissue control material has been available (from New England Nuclear under the tradename Rianen Assay System) and it has failed to relieve the existing interlaboratory confusion resulting from the plethora of distinguishable techniques and methods being practiced. As expected, this nonuniformity of procedures effectively prohibits useful correlation of interlaboratory results.
The lack of common procedures has been further exacerbated because some investigators have sought to maintain a single laboratory control by making their own progesterone or estrogen receptor controls, typically from rat uteri. As may be expected, clinical environments do not lend themselves to the efficient production of controls, especially since production of these materials is a difficult, time consuming and complex matter. Devotion of valuable resources to the production of controls not only reduces the clinical laboratory's effectiveness, but also introduces significant sources of error further limiting the ability to compare results from different laboratories.
It is an object of the present invention to provide a control material possessing both estrogen and progesterone receptors which may be commonly used by laboratories as a control reagent thereby instituting a standard permitting the comparison of laboratory results.
Materials previously employed to provide estrogen receptors include human breast tumor and calf uterus tissue. It has been reported that when such tissue is lyophilized and kept at 0.degree.-4.degree. C., there may be no loss of estrogen binding sites for up to two years. See Benraad, et al., "Estradiol Receptor Activity in Lyophilized Calf Uterus and Human Breast Tumor Tissue", Cancer 46:2762-2764, 1980. It may be noted, however, that the material described by Benraad provides only estrogen binding receptor sites and makes no mention of progesterone binding sites. Progesterone sites have been traditionally characterized as even more unstable than estrogen sites thus greatly increasing the difficulty of providing a stable reagent material having both types of binding sites.
It is another object of the present invention to overcome these problems by providing a material having both progesterone and estrogen receptors in a form having the stability necessary for acceptance in the clinical environment.
Still other sources may be used to provide receptor binding sites. See for instance Korenman, "Radio-Ligand Binding Assay of Specific Estrogens Using a Soluble Uterine Macromolecule", J.Clin.End. and Med. 28:127-130, 1968 which describes the use of uteri from pregnant rats. Bojar, et al., "Investigation of the Thermostability of Steroid Hormone Receptors in Lyophilized Calf Uterine Tissue Powder", Cancer 46:2770-2774, 1980 provides discussion with respect to calf uterine tissue.
The actual structure of the receptors is still a subject for investigation and some recent theories are described by Wittliff in "Steroid Receptor Interactions in Human Breast Carcinoma", Cancer 46,12:2953-2960 (1980). Additional experimental results describing the ability of vanadate and sodium molybdate to inhibit the receptor activation process and thus preserve receptor activity necessary for subsequent assay testing, have been reported in a series of articles: Nishigori, et al., "Inhibition of Progesterone Receptor Activation by Vanadate," archives of Biochem and Biophysics, Volume 203, 2:600-604, 1980; Anderson, et al., "Sodium Molybdate Increases the Amount of Progesterone and Estrogen Receptor Detected in Certain Human Breast Cancer Cytosols," Steroids Volume 35, 3:273-280, 1980; and Maki, et al., "Alterations in Glucocorticoid Receptor Conformation by Molybdate," J. Biochem. 87, 6:1851-1854 (1980).
As has been previously mentioned, investigators in this field have searched for a useful, stabilized cytosol control preparation. Stabilized as used herein means there should be a recovery of at least 90% of the original receptor activity following storage. A cytosol type of preparation, as opposed to the well-known powders heretofore generally available, constitutes tissue which has been homogenized and centrifuged to form a cell-free solution. Although cytosol solutions may be readily prepared, heretofore, none of these cytosol solutions contained both progesterone and estrogen receptors in a stabilized format. For instance, the cytosol preparation described by Vermousek, et al., in "Stable Standard for Determination of Oestrogen Receptor," J.Clin.Chem.Biochem 19:865, 1981, failed to provide more than 20% of the original binding capacity following lyophilization.
It is another object of the instant invention to provide a cytosol preparation suitable for lyophilization and storage in that form for at least a year which, upon reconstitution, provides at least 90% of the original receptor activity, said receptors including both estrogen and progesterone receptors.
The inability of conventional methods to provide such a preparation is further exemplified in an article by Koenders, et al., "Preparation of Lyophilized Reference Samples for Quality Control of Steroid Receptor Measurements," The Ligand Review 3:22-39, 1981. That article describes the stability of lyophilized tissues with respect to estrogen and progesterone receptor activities and at 4.degree. storage, good activity was reported after 14 months. Compare this however to the reported lyophilized cytosol of pig and calf uterine tissues during storage over a similar period of time. As expected, the more hardy ER binding sites remained relatively unchanged; however, PgR activity became virtually nonexistent after only one and a half months of lyophilization storage. Indeed, even lyophilized human breast tumor tissue showed significant decreases in activity after only a few months' storage.
It is yet a further object of the present invention to provide stabilization methods and preparations to permit stabilized storage of calf uterine cystols having both ER/PR binding sites.